Reaction mass of N, N'-hexane-1,6-diylbis [12-hydroxyoctadecanamide] and 12-hydroxy-N-[6-[1-oxoalkyl)amino] hexyl ] octadecanamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 March 2006 20 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): AD-1000
- Substance type: white powder
- Physical state: powder
- Storage condition of test material: at room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: approx. 10-11 weeks old
- Weight at study initiation:
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material.
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 23.1
- Humidity (%): 31 – 58
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0, 5, 10, 25%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A series of two test substance concentrations was tested, selected from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1% and if needed further lower concentrations using the same steps. The highest concentration, selected from this series, was the maximum concentration that could technically be applied.
- Irritation: No irritation was observed in any of the animals examined.
- Lymph node proliferation response: Based on the results, the highest test substance concentration selected for the main study was a 25% concentration.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI  3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM.


TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.
The dorsal surface of both ears was epidermally treated (25 μl/ear) with the test substance concentration, at approximately the same time per day. On the days 1-3, the control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered. On day 6 each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After approximately five hours, all animals were killed by intra peritoneal injection with pentobarbital (0.2 ml/animal). The draining (auricular) lymph node of each ear was excised.
The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 ml PBS.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

none

Results and discussion

Positive control results:

The SI values calculated for the alpha-hexylcinnamic aldehyde concentrations 5, 10 and 25% were 1.6, 3.0 and 5.5 respectively. An EC3 value of 10.0% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 8.8, 5.5, 7.3, 10.3, 9.5 and 8.0%.
Based on these results it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 5, 10 and 25% were 1.5, 1.8 and 1.9, respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 247, 294 and 320, respectively. The mean DPM/animal value for the vehicle control group was 165.

Any other information on results incl. tables

Calculation of stimulation index (SI)

group	treatment	induction	mean DPM ± SEM	SI ± SEM
2	experimental	5% test substance	247 ± 45	1.5 ± 0.3
3	experimental	10% test substance	294 ± 89	1.8 ± 0.4
4	experimental	25% test substance	320 ± 88	1.9 ± 0.4
1	vehicle control	Propylene glycol	165 ± 48	1.0

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.5, 1.8 and 1.9, respectively.
There was no indication that the test substance could elicit a SI >= 3. It was established that the EC3 value (if any) exceeds 25%.
The six-monthly reliability check with alpha-hexylcinnamic aldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.